Plant–plant interactions determine natural restoration of plant biodiversity over time, in a degraded mined land

Abstract Restoration of degraded environments is essential to mitigate adverse impacts of human activities on ecosystems. Plant–plant interactions may provide effective means for restoring degraded arid lands, but little is understood about these impacts. In this regard, we analyzed the effects of two dominant nurse plants (i.e., Artemisia sieberi and Stipa arabica) on taxonomic, functional, and phylogenetic diversity across different ages of land abandonment (i.e., control, recent, and old ages) in a limestone mine site in Iran. In addition, we considered two spatial scales: i) the plot scale (i.e., under 1m2 plots) and ii) the vegetation‐patch scale (i.e., under the canopies of nurse plants), to assess nurse plant effects, land abandonment ages, and their relative importance on biodiversity facets by performing Kruskal–Wallis H test and variation partitioning analysis. Our results indicated an increase in taxonomic, functional, and phylogenetic diversity at the plot scale, when considering the presence of nurse plants under old ages of land abandonment. Such significant differences were consistent with the positive effects of Artemisia patches on taxonomic diversity and Stipa patches on functional and phylogenetic diversity. In addition, we found a larger contribution from nurse plants than land abandonment age on biodiversity variation at both spatial scales studied. Therefore, these results indicate the importance of plant–plant interactions in restoring vegetation, with their effects on the presence of beneficiary species and their functional and phylogenetic relatedness depending on the nurse life forms under the stress‐gradient hypothesis.


| INTRODUC TI ON
Mining limestone for cement is a major industrial activity in at least 15 countries (Mucina et al., 2019;Pratiwi et al., 2021). Today, the number of ecosystems destroyed by these mining activities is increasing, and many studies have shown negative impacts of the presence of post-mining sites on ecosystem health (Palmer et al., 2010).
Therefore, the restoration of degraded sites is essential to mitigate the adverse impacts of mining on the ecosystem (Khater & Arnaud, 2007;Mucina et al., 2019). Restoration is a long process, as affected ecosystems have lost their biodiversity and most of their ecosystem functions and services (Sandell Festin et al., 2019). Ecologists preferably emphasize the re-establishment or increase of biodiversity as a goal of restoration. Biodiversity is typically associated with the increase in ecosystem functions or ecosystem services (Mucina et al., 2019;Pratiwi et al., 2021;Rey Benayas et al., 2008). In this regard, assessing biodiversity and ecosystem services in post-mining sites has received wide attention among restoration scientists (Pitz et al., 2015). However, there is still little understanding of the long-term changes in plant biodiversity and appropriate approaches for enhancing biodiversity in areas degraded after quarrying of limestone and other minerals.
Several approaches and measures are typically used to bring back the lost ecosystems that have suffered from mining (Adesipo et al., 2021;Bradshaw, 2000;Giannini et al., 2016). In recent decades, some conventional techniques such as waste removal, dam building, and on-site containment by sealing have usually been applied for the restoration of mining-affected lands (European Commission, 2009).
However, these methods are criticized for being cost-effective, time-consuming and unacceptable to the general public, and impossible to apply at large scales due to geo-structural and environmental restrictions (Adesipo et al., 2021;Conesa & Schulin, 2010;Tordof et al., 2000). Therefore, restoration ecologists recently suggested plant-based techniques to restore degraded ecosystems in a costeffective and environmentally friendly manner compared with other approaches based on some recent conceptual models hypothesizing plant facilitation as an interaction exploitable in restoration (Cuevas et al., 2013;Navarro-Cano et al., 2017;Ren et al., 2008). In such restoration projects, the key target is usually the re-establishment of the plant community by foundation species such as nurse plants, which ameliorate severe conditions for the recruitment and survival of plant species (Giannini et al., 2016;Ren et al., 2008). For example, several recent studies in degraded ecosystems under mine tailings (Gomez-Aparicio, 2009;Navarro-Cano et al., 2017) or burnt ecosystems (Paniw et al., 2017) have shown important effects of nurse plants in restoring the vegetation depending on the functional traits of nurse and beneficiary species. Although these studies empirically showed that nurse plants affect vegetation restoration by allowing the recruitment and survival of beneficiary species, some challenging issues such as how to change the effects of nurse species depending on stress level, different beneficiary relatedness (i.e., functional or phylogenetic relatedness) and spatial scale are still unclear (Craft, 2016;Jankju, 2013;Padilla & Pugnaire, 2006). The majority of studies have shown important effects of nurse plants on structuring biodiversity facets, including taxonomic, functional, and phylogenetic diversity, through effects on beneficiary relatedness and the promoting of recruitment and survival (e.g., Butterfield & Briggs, 2011;Callaway, 2007;Cavieres et al., 2013;Le Bagousse-Pinguet et al., 2019;Madrigal-Gonzalez et al., 2020;Valiente-Banuet et al., 2007). However, biodiversity facets beneath nurse canopies may or not may exhibit consistent patterns depending on how functional traits conserve across phylogeny, as well as on levels of environmental harshness. For example, different responses of functional and phylogenetic diversity to nurse plant effects could be related to (1) functional traits that are not phylogenetically conserved (Valiente-Banuet & Verdú, 2013;Vega-Alvarez et al., 2019), and (2) functional traits that are labile because they cannot exhibit positive effects of nurse plants on beneficiary relatedness with an increase in environmental severity . In this regard, some meta-synthetic studies (Gomez-Aparicio, 2009;He et al., 2013) show that growth form and some physiological traits such as leaf carbon and nitrogen content may clearly reflect plant strategies in response to major stress factors, including land use, land degradation, and changing soil nature in degraded ecosystems such as limestone mines (Wang et al., 2021). In limestone mines, there is extreme ecological condition in soil by a stronger proportion of calcium carbonate (CaCO3), soluble salts and by its neutrality or even its alkalinity (up to pH = 8.5) that influence plant growth and survival through inhibiting the activity of micro-organisms, impeding the formation and mineralization of humus, reducing the availability of nitrogen, iron, and phosphorus (Kolodziejek & Patykowski, 2015;Nakata, 2015). However, mechanisms promoted by nurse species on plants are not clearly recognized about functional traits related to extreme ecological conditions in degraded ecosystems such as limestone mines. However, the mechanisms promoted by nurse plants in restoring biodiversity are not clearly recognized in relation to such functional traits and stress factors.
Plant-plant interactions specifically influence biodiversity facets and beneficiary relatedness depending on the biodiversity facets and life forms of nurse species in particular (Pashirzad et al., 2019;Soliveres et al., 2012). For example, some studies indicated that cushion plants have the most important effects on taxonomic and phylogenetic diversity in alpine environments Cavieres et al., 2013), whereas nurse grasses can strongly affect functional diversity by providing suitable microhabitats for beneficiary species with distant relatedness (Gastauer et al., 2018;Navarro-Cano et al., 2014). However, nurse plant effects on biodiversity facets may or not may be consistent across different spatial scales. This is particularly problematic, because most inference of nurse plant's effects is based on studies at the patch scale, rather than at the higher spatial scale (Brooker et al., 2008;Soliveres et al., 2012). Moreover, biodiversity patterns are highly scale-dependent and different conclusions about the relative importance of biotic and abiotic factors can result from biodiversity analyses at different spatial scales (Cavender-Bares et al., 2006;Swenson et al., 2006).
Because of these issues, taxonomic, functional, and phylogenetic approaches in restoration ecology should be applied across different spatial scales to enhance the entire restoration process (Hipp et al., 2015;Pitz et al., 2015). However, such a framework has been rarely applied when studying the restoration of degraded ecosystems (Ren et al., 2008;Singh et al., 2012). We might, therefore, better understand the effects of nurse plants on the restoration of ecosystems by considering the features of beneficiary species beneath nurse plants with different life forms, their relationships at different ages of abandonment since the cessation of mining and at different spatial scales (Gastauer et al., 2013;Perring et al., 2015). The availability of restored sites at different ages of abandonment and different spatial scales will enable a field observation of chronosequence changes in vegetation regarding effects of plantplant interactions for land restoration (Cadotte, 2013;Clark et al., 2012;Navarro-Cano et al., 2014;Prach & Tolvanen., 2016).
Here, we analyzed the effects of plant-plant interactions on taxonomic, functional, and phylogenetic diversity at the plot and vegetationpatch scales with respect to different ages of abandonment in a limestone mine located in northeastern Iran. Previous studies only addressed either the effects of abandonment age or the presence of nurse plants on biodiversity, especially taxonomic diversity .

| Study area
Our study region consists of abandoned mine lands and nearby natural rangelands in Cement-Shargh, northeastern Iran (centered around 59.7467 N, 36.4746 E). Elevation ranges from 1000 to 1180 m, generally increasing from south to north (Table 1; Figure S1). Facilitative effects of Artemisia species on understory plant species were clearly observed especially under moderate environmental conditions (Jankju, 2013;Jankju & Ejtehadi, 2016). Artemisia species generally create suitable microenvironments for understory species by increasing soil moisture and decreasing the irradiation and air temperature compared with open areas (Jankju et al., 2008).
However, allelopathic effects have been observed on seed germination and seedling establishment, and this may influence total species diversity (Jankju, 2013). S. arabica is a herbaceous perennial bunchgrass that can grow to 0.39 m and has facilitative effects on the growth, recruitment, and survival of other species (Jankju, 2013;Jankju et al., 2008;.

| Site selection
We selected three sites based on different ages of abandonment since mining. They included control sites with relatively no mining activities and natural vegetation, old abandoned sites with mining activities terminated 50 or more years ago, and recently abandoned sites with mining having occurred within the last 10 years (Table 1; TA B L E 1 Three sites were used in this study with respect to different ages of abandonment since mining activities (recent abandonment, old abandonment, and control sites with relatively no mining activities) in Cement-Shargh in northeastern Iran. All the sites were assessed in two different scales of vegetation (i.e., plot scale including 1 × 1 m 2 plots with and without the presence of nurse species and patch scale considering vegetation beneath Artemisia sieberi and Stipa arabica and their open areas with 0.5 × 0.5 m 2 plots) see details in Appendix S1). Importantly, we selected sites with the least impact from cement pollution to reduce bias in our results. To examine abandonment age, we extracted the time of last mining activity for each site from aerial photographs and from information on mining time recorded by staffs at the Cement-Shargh site.

| Vegetation sampling based on nurse plant effects and land abandonment ages
To evaluate nurse plant effects with respect to land abandonment ages (i.e., across three sites), we considered two spatial scales: (1)  Open patch) (see details in Appendix S1). To properly assess the nurse plant effects across different spatial scales, it is important that the vegetation-patch scale is located within larger spatial scale (Pashirzad et al., 2019;Soliveres et al., 2012). The number of samples and patch cover (i.e., 0.5 × 0.5 m) at the vegetation-patch scale were obtained through assessing species-area curves and previous groundbreaking references that advise a range of 20 to 30 samples with 0.5 × 0.5 m cover beneath the canopies of nurse species as appropriate replication and sample size for vegetation sampling beneath grasses and dwarf shrubs (Soliveres et al., 2012
In addition, these traits were introduced as key functional traits for plant species in degraded environments and are dependent on biotic interactions, environmental conditions, and human-based disturbances (Lavorel, 2013;Lienin & Kleyer, 2012;Meng et al., 2015;. Leaf dry matter content (LDMC), specific leaf area (SLA), leaf nitrogen, and carbon content are related to growth rate and litter quality (Cortez et al., 2007;Dechaine et al., 2014;Kazakou et al., 2006). Plant height, growth form, and seed mass are strongly related to the ability of plants to compete, demographic features such as longevity and survival, respectively (Cornelissen et al., 2003;Moles et al., 2009;Ostertag et al., 2014). Life span represents plant strategies and abilities including competitive or facilitative abilities (Rahmanian et al., 2019).
We obtained information on these plant functional traits from publicly available trait datasets (BIEN package in R (Maitner et al., 2018), TRY (Kattge et al., 2011), LEDA (Kleyer et al., 2008) and TR8 (Bocci, 2014). Functional trait information was more obtained from BIEN package than other publicly available trait databases (because trait information for plant species in majority of worldwide is more available in BIEN than other databases such as TRY and LEDA).
In addition, we considered plant height based on the vegetative part, because reproductive parts in many plant species especially in grasses create some biased measurements based on their variations in different growth stages

| Phylogenetic information
We obtained a phylogeny of 37 plant species present in all studied sites based on the most up-to-date megaphylogeny for seed plants (Smith et al., 2018). We standardized the species names in our dataset according to The Plant List using the R package "Taxonstand" (Cayuela et al., 2012). Then, we used the R function V. PhyloMaker (Jin & Qian, 2019) to link the species names in our dataset with those in the megaphylogeny, and the scenario 3 approach (Qian et al., 2016), to add species to the phylogeny. This phylogenetic tree was then used as reference lists from which phylogenetic diversity could be calculated for our communities and pairwise microsites in the dataset.

| Measures of species composition and plant functional groups
To measure the species composition, we performed detrended cor- This index in the abundance-weighted case is equivalent to Rao's Q and Hill numbers (Webb et al., 2002). Before measuring the functional diversity, we used Pearson correlation analysis to analyze the correlation between the traits. We did not find significant correlations between functional traits studied (see details in Appendix S3).
Therefore, we considered all these functional traits in

| Statistical analyses
We analyzed variation in all biodiversity facets relative to i) the presence of nurse plants and abandonment age at plot scale and ii) different patches (i.e., Stipa patch and Artemisia patch) and abandonment age at vegetation-patch scale. Differences in biodiversity indices were calculated across all studied sites. To measure differences in plant biodiversity, we used Kruskal-Wallis tests followed by Dunn's post hoc tests with Bonferroni correction (Haselberger et al., 2021) by subtracting control sites from sites in different ages of abandonment and comparing across all studied sites with available data (Haselberger et al., 2021).

| Species composition and plant functional groups under sites with different ages of land abandonment
Species composition and plant functional groups differed significantly between sites with different land abandonment ages (Figure 1).
In old abandoned sites, we found a wide range of plant species (

| Effects of plant-plant interactions and abandonment ages on biodiversity facets at the plot scale
Taxonomic diversity indices at the plot scale responded significantly to the presence of nurse plants and land abandonment age

| Effects of plant-plant interactions and abandonment ages on biodiversity facets at the vegetation-patch scale
At the vegetation-patch scale, all biodiversity facets differed with abandonment age and nurse plant life form. Generally, taxonomic, functional, and phylogenetic diversity in sites with Stipa patches differed from sites with Artemisia patches (Figures 3, 4). In this re-

| The contributions of study factors on biodiversity variation at different spatial scales
The amount of variance explained for all diversity facets increased when considering nurse species at both spatial scales (pink fraction in

| DISCUSS ION
Biodiversity facets at the plot scale differed significantly with the presence of nurse plants across ages of land abandonment.
In this regard, a significant increase in taxonomic and functional/ phylogenetic diversity was observed, when considering plots with nurse species than plots without nurse species. Such differences in taxonomic and functional/phylogenetic diversity were consistent with biodiversity patterns beneath Artemisia and Stipa patches. In this regard, dominant shrub (i.e., A. sieberi) strongly F I G U R E 2 Boxplots showing differences in taxonomic (i.e., species richness (q0) and Shannon's diversity) (q1), functional (FSES.mpd) and phylogenetic (PSES.mpd) diversity at plot scale among three studied sites with respect to the presence (blue box; n = 20 1 m 2 plots in each study site) or absence (red box; n = 20 1 m 2 plots in each study site) of nurse species and different ages of abandonment. Box edges indicate the 25th-75th percentile for each variable. Error bars indicate 5th and 95th percentiles. Significant differences across all studied sites and between studied sites with respect to abandonment age after mining activities are reported from Kruskal-Wallis tests followed by Dunn's post hoc tests with Bonferroni correction. In addition, significant differences between plots with respect to the presence of nurse species are reported from the Wilcoxon-Mann-Whitney test as *p < .05, **p < .01, and ***p < .001 recent abandoned site). Therefore, we strongly confirm that positive impacts of nurse species are significantly associated with environmental or disturbance level considered Soliveres et al., 2015).

| Effects of nurse types on restoration of biodiversity facets
The effects of different patches on specific biodiversity facets suggest that there are likely impacts of the morphological features of nurse plants, as well as some processes controlling beneficiary diversity, on the restoration of plant biodiversity (Soliveres et al., 2012;Valiente-Banuet & Verdu, 2013). A significant increase in taxonomic diversity beneath Artemisia patches (see q0 and q1 in Figure 2) under moderate disturbance condition could be a consequence of specific shrub canopy characteristics on beneficiary species. A. sieberi shrubs have large canopy areas, which may provide a large suitable microhabitat to facilitate a large number of beneficiary species (Jankju et al., 2013). Additionally, the porous canopy of A.
sieberi may promote greater spatial segregation between beneficiary species (Pistón et al., 2016), reducing competition between them.
Such spatial segregation between understory species may allow greater coexistence without the need for strong trait or evolutionary differences (Losapio et al., 2021). Our results provided strong support for these explanations, illustrating an increase in taxonomic diversity and a decrease in functional/phylogenetic relatedness between plant species in Artemisia patches. In addition, our findings and some previous studies (Jankju et al., 2008;Perring et al., 2015) confirm that allelopathic effects of Artemisia plant in one successional stage may facilitate the presence of plants in the next successional stage. In this regard, although there is an explanation about excluding some plant species via Artemisia's allelopathic effects (Jankju, 2013;Rahmanian et al., 2021), a vast suitable area beneath Artemisia due to niche partitioning will provide an increase in species richness . However, niche partitioning in the Artemisia patches in our study was only observed under moderate disturbance conditions. In this regard, there is a strong support for stress-gradient hypothesis that states some morphological and allelopathic features of Artemisia patches are significantly observed under moderate conditions (Aerts et al., 1991;Jankju, 2013;Köchy & Wilson, 2000;Luiz et al., 2013;Pistón et al., 2016).
An increase in functional/phylogenetic diversity under Stipa patches may be caused by negative interactions between beneficiary species (Goldberg et al., 2001;Winkler et al., 2014). Grasses generally provide a specific microhabitat based on their competitive nature, surface roots, dense and small canopy area (Craft, 2016;Padilla & Pugnaire, 2006). In such microhabitat, negative interactions such as competition will occur between beneficiary species. In addition, nurse grasses also compete efficiently with beneficiary species based on their morphological features, including fibrous roots and a large root: shoot ratio (Gomez-Aparicio, 2009;Zhenqi et al., 2012).
Such negative interactions may lead to niche divergence among beneficiary species by enhancing the degree of overdispersion among traits and so decreasing evolutionary relatedness (Butterfield & Briggs, 2011;Gastauer et al., 2018;Gavini et al., 2019). Thus, fewer, more functionally or phylogenetically divergent, species may be able to coexist through interactions between nurse grasses with beneficiary species (Navarro-Cano et al., 2017) (Figure 3; a decrease in taxonomic diversity and an increase in functional/phylogenetic diversity are illustrated in Stipa patches). However, such competition between interacting species will significantly occur when disturbance conditions are moderate (i.e., under old abandoned site that plant species with different functional identities such as herbs and grasses with different SLA and plant height were observed) (Soliveres et al., 2012. This explanation is consistent with our variation partitioning results, which suggest important impacts of land abandonment age and the presence of nurse grasses on plant biodiversity (Butterfield & Briggs, 2011;Gavini et al., 2019).

| Conservation and management strategies based on our new and novel findings
Our study provided some new and novel results regarding the im-

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting this study are available at Appendix S4 in Supporting Information file.